Lung cancer therapy using an engineered respiratory syncytial virus

ABSTRACT

The invention discloses an engineered oncolytic respiratory syncytial virus (RSV), NS1 gene deficient RSV, and its usage to treat lung cancer by killing cancer cells with in vitro and in vivo evidences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit, as a divisional, of U.S. applicationSer. No. 12/925,886, filed Nov. 2, 2010, which claims priority to U.S.non-provisional application Ser. No. 12/800,585, filed on May 18, 2010,and U.S. provisional Application No. 61/398,236, filed on Jun. 22, 2010,the disclosures of which are incorporated herein in their entirety byreference.

DESCRIPTION

1. Field of the Invention

The invention is within the scope of oncolytic virotherapy. Weengineered respiratory syncytial virus (RSV) by deleting NS1 gene, andfound that the NS1 gene deficient-RSV (ΔNS1 RSV) can kill lung cancercells, but not normal human cells.

2. Background of the Invention

Lung cancer: treatment and survival. Lung cancers are divided byhistopathology into small cell lung cancers (˜15%) and NSCLC (˜85%) [1].In 2009, 219,440 new cases are expected and 159,390 persons areprojected to die from lung cancer in the United States [2]. Prevailingtreatments have only limited success in lung cancer, particularly NSCLC,which becomes resistant to the drugs used for chemotherapy.

Radiotherapy, alone or in combination with surgery or chemotherapy, isuseful in the management of NSCLC [3]. However, tumor radio-resistance,including intrinsic radio-resistance before treatments and acquiredradio-resistance during radiotherapy, makes radiotherapy problematic forNSCLC [4]. There is no effective treatment available for advanced ormetastatic NSCLC [5]. The global increase in lung cancer, together withits poor survival rate and resistance to classical chemotherapy,underscores the need for development of novel therapeutic strategies.

Oncolytic virotherapy. Oncolytic virotherapy is a novel strategy usingviruses, either naturally occurring or genetically modified, toselectively target and destroy tumor cells while leaving surroundingnon-malignant cells unharmed [6]. Our preliminary data show that ΔNS1RSV replicates to a high titer in lung tumor cells, compared to thenormal WI-38 diploid lung cells (FIG. 2B), and ΔNS1 RSV, not wt RSV,specifically kills lung cancer cells, but not normal WI-38 or NHBE cells(FIGS. 2A and 4A and Table 1). NS1 protein functions as ananti-apoptotic factor (FIG. 4A, B) and deletion of NS1 restores theapoptotic pathway in tumor cells.

TABLE 1 ΔNS1 RSV preferentially kills human lung cancer cells. Virus(MOI = 10) ΔNS1 RSV wt RSV Cells CPE (24 h post-infection) WI-38 cells(Human normal embryonic lung − − fibrolast) NHBE cells (Normal humanbronchial epithelial) − − H157 cells (erlotinib-resistant) ++++ − H480cells (erlotinib- and dasatinib-resistant) ++++ − H1299 cells(erlotinib-resistant and p53−/−) ++++ − H441 cells (erlotinib- anddasatinib-resistant) +++ − H368 cells +++ − H1335 cells ++++ − A549cells (erlotinib-resistant, dasatinib- ++++ − partially resistant) H23cells (erlotinib- and dasatinib-resistant) +++ − Note: −: no CPE; +++:CPE 50%-75%; ++++: CPE >75%

Biology of RSV NS1 protein. RSV genome contains individual genes for tenviral proteins [7]. The transcription of RSV genes is polar, with thepromoter-proximal genes being transcribed more frequently than thepromoter-distal ones. The NS1 gene is promoter-proximally located at the3′ end of the viral genome and therefore its mRNA is the most abundantof the RSV transcripts in a linear start-stop-restart mode [8] (FIG. 1).NS1 protein is referred to as nonstructural since it has not beendetected in RSV particles. NS1 is exclusively found in RSV-infectedcells. Our group, along with others, has found that NS1 can counter thetype I IFN signaling during RSV infection [9, 10], implying that NS1plays a direct role in inhibiting the host's innate immune response.

Mitochondria as targets for anticancer agents. Evasion from apoptoticcell death unregulated cell proliferation and eventual tumor developmentis one of the hallmarks of oncogenic cell transformation. We found thatΔNS1 RSV selectively induces apoptosis in tumor cells (FIG. 4), and alsodecreases mitochondrial ΔΨm and promotes mitochondrial swelling in A549lung cancer cells, suggesting that mitochondrially-mediated apoptosisparticipates in the anti-tumor effect of ΔNS1 RSV.

RSV can be rendered nonpathogenic by mutating the NS1 gene so that it nolonger inhibits IFN release, which attenuates viral infection in normalcells. However, these nonpathogenic RSV, ΔNS1 RSV, are still oncolyticbecause tumor cells are defective in their ability to produce andrespond to IFN and, therefore, efficiently support the propagation ofΔNS1 RSV.

SUMMARY

This invention discloses a NS1 gene-deficient RSV (ΔNS1 RSV), whichcould be utilize to kill lung cancer cells, but not normal human cells.In one embodiment, the gene NS1 is deleted by the removal of 122 to 630nt in the antigenomic cDNA using reverse genetics approach, resulting inthe joining of the upstream nontranslated region of NS1 to thetranslational initiation codon of NS2. The ΔNS1 RSV was recoveredthrough co-transfecting Vero cells with the NS1-deficient RSV cDNA andexpressional plasmids encoding N, P, M2-1 and L. The RSV NS1 proteinfunctions as a type-I-IFN antagonist, ΔNS1 RSV virotherapy produces moretype-I-IFN, which prevents virus from replication in normal cells andalso induces antitumor effects

In another embodiment, the engineered virus could be any other virushaving a similar strategy to delete NS1 gene, which functions as a geneencoding the related protein as a type-I-IFN antagonist.

In another embodiment, the ΔNS1 RSV can be applied to cancer spot bydirect injection. Or the ΔNS1 RSV can be delivered to cancer spotthrough blood transfusion.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Diagram of the RSV genome and its transcription and replicationproducts. The virus genes are depicted as grey rectangles; the L gene,which comprises almost half of the genome, has been truncated. The GSand GE signals are shown as white and black boxes, respectively. Theencoded anti-genome and mRNAs are indicated by hatched rectangles.Arrows indicate the location of the promoters.

FIG. 2 A-B. Virus infection of NSCLC and normal cells. (A) Morphology ofvirus-infected A549 and WI-38 cells 24 h post-infection. (B) Viraltiters as measured by plaque assay at 20 h after infection. Standarddeviations from three independent experiments are shown by the errorbars.

FIG. 2 C-D. Subcutaneous A549 tumors were implanted in BALB/c nude miceand virotherapy is indicated by arrows below the x-axis. Control micereceived equal volume of vehicle. Tumor sizes were measured at the endof treatment. Each data point represents a mean of 4 tumors measurementsplus or minus the standard deviation.

FIG. 2 E-F. Tumors and organs were removed from the mice three daysafter injection of viruses. Homogenates of the tissue were prepared andassayed for viral titers and total RNAs were analyzed by RT-PCR forviral F gene expression [3]

FIG. 3. Flow cytometric analysis of cell cycle. A549 cells were infectedwith indicated viruses (MOI=5), and 24 hr later the cells were collectedand fixed with 70% cold aqueous ethanol. Cells were then stained with PIstaining solution that contained propidium iodide (50 μg/ml; Sigma) andRNase A (50 μg/ml; Sigma) in the dark at room temperature for 30 min.Ten thousand cells were measured per sample, and the analysis wasperformed using the Cell Quest Pro software (Becton-Dickinson).

FIG. 4. NS1 protein prevents apoptosis and loss of mitochondrial ΔΨm.(A) A549 cells and NHBE cells were infected with indicated viruses(MOI=5), and collected at 20 and 48 hr post-infection for apoptosisanalysis by annexin V-binding and PI uptake assay. (B-C) A549 cell werecollected at 5 and 20 hr post-infection, and whole-cell lysates wereimmunoblotted. (D) H1299 (p53−/−) cells were treated and apoptosis wasmeasured as described in (A).

FIG. 5. ΔNS1 RSV infection decreases mitochondrial ΔΨm and inducesmitochondrial ultrastructural alterations in A549 cells. (A-B) JC-1 dyewas used to measure changes in mitochondrial ΔΨm by flow cytometryfollowing viral infection. The red:green fluorescence intensity of JC-1gives an index of the ΔΨm. P≦0.05 between wt and ΔNS1 RSV. (C)Transmission electron microscopy of mitochondrial morphology ofvirus-infected A549 cells is characterized by electron-dense matrix(arrows) and size of mitochondria.

Table 1. Cytopathic effect (CPE) test showing ANSI RSV selectively killslung cancer cells

DETAILED DESCRIPTION OF THE INVENTION

The respiratory syncytial virus (RSV) was used in this study. The NS1gene was deleted by the removal of 122 to 630 nt in the antigenomic cDNAusing reverse genetics approach, resulting in the joining of theupstream nontranslated region of NS1 to the translational initiationcodon of NS2. The ΔNS1 RSV was recovered through co-transfecting Verocells with the NS1-deficient viral cDNA clone and expressional plasmidsencoding N, P, M2-1 and L. Alternatively, the engineered virus could beany other viruses with the deletion of similar NS1 gene.

ΔNS1 RSV preferentially kills NSCLC cells both in vitro and in vivo.NSCLC cells and WI-38 normal human diploid lung cells were infected withwt or ΔNS1 RSV (MOI=5). Changes in cell morphology were observed andviral replication was measured. FIG. 2A shows that ΔNS1 RSV selectivelyinduces CPE in A549 cells, and that ΔNS1 RSV has a higher viral titer inA549 cells than in WI-38 cells 24 hr after infection (FIG. 2B),suggesting that A549 cells efficiently support the propagation of ΔNS1RSV because they are defective in producing and responding to IFN. Dr.Bose's group reported that wt RSV kills prostate cancer cells [11]. Totest if the higher titer of wt RSV also kills lung tumor cells, weinfected different lung tumor cell lines and normal WI-38 and NHBE cellswith high dose of viruses (MOI=10), and checked CPE 24 hrpost-infection. As shown in Table 1, ΔNS1 RSV, but not wt RSV,preferentially kills NSCLC cells. These experiments were done on celllines in vitro, but proof of efficacy requires demonstration in vivo. Todetermine whether ΔNS1 RSV infection induces tumor growth regression invivo, A549 cells were injected s.c. into the left and right flanks of4-6 weeks old nude BALB/c mice (n=4 per group) and the resulting tumorswere allowed to develop.

Viruses were locally injected into the tumors three times and the sizesof the tumors were measured using digital calipers. FIG. 2C, D show thatΔNS1 RSV infection caused regression in tumor growth versus controls. Totest the safety of locally administered viruses, the virus titer invarious organs of infected mice was determined by plaque assay andRT-PCR assay. As shown in FIG. 2E, F, the viruses specifically localizeto tumors.

ΔNS1 RSV induces sub-G1 peak in A549 cells. Cell cycle dysregulation isa critical feature of tumor cells. The inhibition of cell cycle is apotential therapeutic target for the control of tumor cellproliferation. To test whether ΔNS1 RSV induces cell cycle arrest, weinfected A549 cells with the indicated viruses at an MOI of 5. Analysisof propidium iodide (PI) staining by flow cytometry clearly revealedthat virus infection did not significantly affect tumor cell cycle, butthe appearance of a sub-G1 (apoptosis) peak was considerably elevated inΔNS1 RSV-infected cells (FIG. 3).

ΔNS1 RSV infection induces apoptosis in tumor cells, but not in normalhuman bronchial epithelial cells. To test the differential effect ofΔNS1 RSV infection on apoptosis, A549 cells and NHBE cells were infectedwith the indicated viruses (MOI=5) and apoptosis was measured by theannexin V binding assay. FIG. 4A shows that ΔNS1 RSV selectively inducesapoptosis in tumor cells, compared to the cell spontaneous apoptosisshown in controls, which was verified by immunoblotting (FIG. 4B, C).

Recent research reports demonstrated that p53 participates inRSV-induced apoptosis [12]. To determine if p53 is required for ΔNS1RSV-induced apoptosis, p53-deficient NSCLC H1299 were tested. FIG. 4Dshows that ΔNS1 RSV infection induced apoptosis in H1299 cells,indicating that p53 protein is not an exclusive factor required fordevelopment of apoptosis in ΔNS1 RSV-infected tumor cells.

ΔNS1 RSV infection decreases mitochondrial ΔΨm and causes mitochondrialswelling. We found that ΔNS1 RSV triggered apoptosis in lung cancercells through mitochondrial pathway (FIG. 4B-C). To test the effects ofviral infection on mitochondrial ΔΨm, we measured mitochondrial ΔΨm inA549 cells upon viral exposure (MOI=5). As shown in FIG. 5A, B, NS1prevented loss of mitochondrial ΔΨm in response to viral infection. Wefurther confirmed mitochondrial ΔΨm results by transmission electronmicroscopy. Mitochondria in vehicle-treated A549 cells exhibit acharacteristic electron-dense matrix, in contrast to the swollenmitochondria with a loss of electron density in the matrix of ΔNS1RSV-infected cells. Cells infected with wt RSV show less mitochondrialalteration than ΔNS1 RSV-infected cells. IFN-β did not significantlyaffect mitochondrial morphology upon ΔNS1 RSV infection (FIG. 5C).

REFERENCES

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1. A method of treating a neoplasm in a subject, comprisingadministering to the subject an oncolytically effective amount of arespiratory syncytial virus (RSV) with the NS1 gene deleted or mutatedso that it is no longer functional, thereby treating the neoplasm in thesubject.
 2. The method of claim 1 where the RSV NS1 gene is deleted. 3.The method of claim 2 where the RSV NS1 gene is deleted by a reversegenetics approach.
 4. The method of claim 1 where the neoplasm is a lungcancer.
 5. A method of treating lung cancer comprising resuspending anoncolytic respiratory syncytial virus (RSV) with the NS1 gene deleted ormutated so that it is no longer functional in saline or medium, andinjecting the RSV suspension into cancerous tissue or intravenously. 6.The method of claim 5 where the RSV NS1 gene is deleted.
 7. The methodof claim 6 where the RSV NS1 gene is deleted by a reverse geneticsstrategy.
 8. The method of claim 5 where the RSV NS1 gene is mutated sothat it is no longer functional.
 9. The method of claim 5 wherein theRSV further comprises viral NS2, N, M, SH, G, F, M2-1, P, and L genes.